High doses of dietary zinc induce cytokines, chemokines, and apoptosis in reproductive tissues during regression

In chickens, high levels of dietary zinc cause molting, and the reproductive system undergoes complete remodeling concomitant to feather replacement. In the present study, the expression profiles of cytokines and chemokines were investigated in the ovary and oviduct of control hens and of hens induced to molt by zinc feeding. The zinc-induced feed-intake suppression, the changes in corticosterone levels, the immune cell populations in the reproductive tract, and the apoptosis of reproductive tissues were analyzed. The expression of mRNAs for interleukin-6 (IL-6), interferon-γ (IFN-γ), the avian ortholog of mammalian IL-8 (chCXCLi2), and a chicken MIP-1β-like chemokine (chCCLi2) in the ovary and of mRNAs for IL-1β, IL-6, IFN-γ, transforming growth factor-β2, chCXCLi2, and chCCLi2 in the oviduct were upregulated significantly during zinc-induced molting. A simultaneous feed-intake reduction was observed with higher expression of cytokines and chemokines. The results of the present investigation also suggested that the upregulation of corticosterone was closely associated with the increased expression of cytokines and chemokines. An increase in apoptosis within reproductive tissue during tissue regression was also noted. We had previously observed the upregulation of these cytokines expression in an earlier study (molting by feed withdrawal). However, the pattern and the level of expression were different among these two methods. These findings indicate that cytokines might be a common mediator of tissue regression during molting induced by diverse methods, although the pattern of induction is different. Thus, a high dose of dietary zinc seems to induce reproductive regression via the upregulation of cytokines and chemokines, the suppression of feed intake, and the increase in serum corticosterone, resulting finally in the apoptosis of reproductive tissues.     

Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary

The gonadal development of chicken embryo is regulated by hormones and growth factors. Transforming growth factor beta (TGF-β) isoforms may play a critical role in the regulation of growth in chicken gonads. We have investigated the effect of the TGF-β isoforms on the number of germ and somatic cells in the ovary of the chicken embryo. Ovaries were obtained from chicken embryos at 9 days of incubation. They were organ-cultured for 72 h in groups treated with TGF-β1, TGF-β2, soluble betaglycan, TGF-β1 plus soluble betaglycan, or TGF-β2 plus soluble betaglycan, and untreated (control). TGF-β1 and TGF-β2 diminished the somatic cell number in the ovary of the chicken embryo at this age by inhibiting the proliferation of the somatic cells without increasing apoptosis. On the other hand, TGF-β1 and TGF-β2 did not affect the number of germ cells in the cultured ovary. The capacity of TGF-β1 and TGF-β2 to diminish the number of somatic cells in the ovary was blocked with soluble betaglycan, a natural TGF-β antagonist. However, changes in the location of germ cells within the ovary suggested that TGF-β promoted the migration of the germ cells from the ovarian cortex to the medulla. Thus, TGF-β affects germ and somatic cells in the ovary of the 9-day-old chicken embryo and inhibits the proliferation of somatic cells.This work was supported by DGAPA-UNAM (IN214403) and CONACYT (45030).     

Immune complex-trapping cells in the spleen of the chicken

Summary By means of immunohistoperoxidase techniques and the use of HRP-anti-HRP complexes, follicular dendritic cells in chicken spleen can be characterized both at the light-microscopical and ultrastructural level. In contrast to findings in mammals follicular dendritic cells in chicken spleen exhibit evident acid-phosphatase activity and possess considerable numbers of primary lysosomes. After intravenous injection of immune complexes a transient immune complex-trapping occurs in the peripheral parts of the Schweigger-Seidel sheath. The immune complextrapping cells in the Schweigger-Seidel sheath and germinal centre show an identical enzyme histochemical pattern and only minor differences in ultrastructural characteristics.Shortly after intravenous injection of immune complexes and carbon particles these compounds show an identical distribution pattern; however, in the following days these distribution patterns become divergent.     

Antigen-driven selection in germinal centers as reflected by the shape characteristics of immunoglobulin gene lineage trees: a large-scale simulation study

During the immune response, the generation of memory B lymphocytes in germinal centers involves affinity maturation of the cells’ antigen receptors, based on somatic hypermutation of receptor genes and antigen-driven selection of the resulting mutants. Affinity maturation is vital for immune protection, and is the basis of humoral immune learning and memory. Lineage trees of somatically hypermutated immunoglobulin genes often serve to qualitatively illustrate claims concerning the dynamics of affinity maturation in germinal centers. Here, we derive the quantitative relationships between parameters characterizing affinity maturation dynamics (proliferation, differentiation and mutation rates, initial affinity of the Ig to the antigen, and selection thresholds) and the mathematical properties of lineage trees, using a computer simulation which combines mathematical models for all mature B cell populations, stochastic models of hypermutation and selection, lineage tree generation and measurement of graphical tree characteristics. We identified seven key lineage tree properties, and found correlations of these with initial clone affinity and with the selection threshold. These two parameters were found to be the main factors affecting lineage tree shapes in both primary and secondary response trees. The results also confirm that recycling from centrocytes back to centroblasts is highly likely.     

Detailed analysis of formation of chicken pituitary primordium in early embryonic development

The primordium of the mammalian adenohypophysis derived from Rathke's pouch (RP) is known to be formed by oral ectoderm invagination. However, in the early phase of pituitary development, the detailed process by which the oral ectoderm develops into the adenohypophysis remains largely unknown. Using high-resolution non-radiolabeled in situ hybridization and the BrdU and TUNEL methods, we have examined the detailed expression pattern of factors involved in the formation of RP of chicken and the changes in the mitotic and apoptotic cell regions in RP. In the chicken embryo, Sonic hedgehog (Shh) mRNA was initially expressed in the stomodeal plate but not in the oral ectoderm. After prospective diencephalon had detached from the oral ectoderm, another Shh-expressing region appeared in the most rostral part of the recess. LIM homeobox gene 3 (Lhx3) mRNA first appeared in the anterior area of Rathke's recess, and expression then spread to the caudal region. alphaGSU mRNA-expressing cells were observed at both ends of the Lhx3-expressing region, and thereafter the expression area moved to the posterior region. Furthermore, a close overlap was found between the proliferating region and Lhx3 mRNA-expressing area, and TUNEL-positive cells appeared in Seessel's pouch derived from the foregut. Thus, the primordium of the pituitary gland corresponding to the Lhx3-expressing region is surrounded by the Shh-expressing region, which appears in two steps, and the mass growth and invagination of RP of chicken result from the coordination of the dorsal extension of the anterior region and the ventral extension of the posterior region of RP.     

Cofilin phosphorylation and actin polymerization by NRK/NESK,a member of the germinal center kinase family

Nck-interacting kinase (NIK)-related kinase (NRK)/NIK-like embryo-specific kinase (NESK) is a protein kinase that belongs to the germinal center kinase family, and activates the c-Jun N-terminal kinase (JNK) signaling pathway. In this study, we examined the effect of NRK/NESK on actin cytoskeletal organization. Overexpression of NRK/NESK in COS7 cells induced accumulation of polymerized actin at the perinuclear. Phosphorylation of cofilin, an actin-depolymerizing factor, was increased in NRK/NESK-expressing HEK 293T cells. In addition, in vitro phosphorylation of cofilin was observed on NRK/NESK immunoprecipitates from HEK 293T cells expressing the kinase domain of NRK/NESK. The cofilin phosphorylation occurred at the serine residue of position 3 (Ser-3). Since the phosphorylation at Ser-3 inactivates the actin-depolymerizing activity of cofilin, these results suggest that NRK/NESK induces actin polymerization through cofilin phosphorylation. The cofilin phosphorylation did not appear to be mediated through activation of LIM-kinasel, a cofilin-phosphorylating kinase, or through the activation of JNK. Thus, cofilin is likely to be a direct substrate of NRK/NESK. NRK/NESK is predominantly expressed in skeletal muscle during the late stages of mouse embryogenesis. Thus, NRK/NESK may be involved in the regulation of actin cytoskeletal organization in skeletal muscle cells through cofilin phosphorylation.     

Immunohistochemical examination of intraspinal serotonin neurons and fibers in the chicken lumbar spinal cord and coexistence with Leu-enkephalin

Intraspinal serotonin-positive cells and fibers were examined in the chicken lumbar spinal cord following removal of descending serotonin fibers by spinal transection. Co-localization of Leu-enkephalin immunoreactivity in intraspinal serotonin cells was also examined using a double immunofluorescence labeling technique. By one or two weeks after spinal transection, virtually all supraspinal serotonin fibers were eliminated. Intraspinal serotonin cells were located ventral or ventrolateral to the central canal corresponding to laminae VII, VIII, and IX, and the anterior funiculus. Intraspinal serotonin cells sent fibers to (1) the pia mater on the ventral or ventrolateral surface of the spinal cord; (2) vessels in the spinal cord; (3) sympathetic preganglionic column of Terni; (4) other intraspinal serotonin neurons; (5) the central canal. Some 30%–50% of the intraspinal serotonin cells co-localized with Leu-enkephalin. Intraspinal serotonin fibers co-containing Leu-enkephalin were observed in the pia mater located on the most lateral surface of the spinal cord. Permanent address: This study was supported by Grant-in-Aid for Scientific Research on Priority Area from the Ministry of Education, Science and Culture, Japan.     

In vivo effects of vascular endothelial growth factor on the chicken chorioallantoic membrane

The effect of vascular endothelial growth factor (VEGF₁₆₅) on the chorioallantoic membrane (CAM) of 13-day-old chick embryos was studied. The factor was applied in doses of 0.5–4 g for a period of up to 4 days. Macroscopical, histological and immunohistological studies were carried out. The localization of the factor was examined with an anti-VEGF antibody. The mitogenicity of VEGF₁₆₅ and basic fibroblast growth factor (bFGF) were studied by means of the BrdU-anti-BrdU method. Furthermore, the effect of heparin alone and in combination with VEGF₁₆₅ was investigated. VEGF₁₆₅ specifically induces angiogenesis in doses of 0.5 g and more. A brush-like formation of blood vessels can be seen in the region of the precapillary vessels. Angiogenesis also takes place in the region of the capillaries and the venules. Histologically we found indications of sprouting as well as of intussusceptive capillary growth. The presence of the factor in the application area could be demonstrated with the anti-VEGF antibody for a period of 3 days. The factor is located in the chorionic epithelium and the intraepithelial capillaries. The BrdU-studies show that VEGF₁₆₅ induces strong endothelial cell proliferation, whereas bFGF elicits fibrocyte proliferation and minor endothelial cell proliferation. Heparin induces squamous metaplasia of the chorionic and allantoic epithelium in combination with an aggregation of fibrocytes. We could not detect any enhancement of VEGF₁₆₅ by heparin.This paper is dedicated to Professor R. Ortmann on the occasion of his 80^th birthday     

Distribution of MAP1A,MAP1B,and MAP2A&;B during layer formation in the optic tectum of developing chick embryos

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M⁺ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B⁺ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A^–/MAP2A&B^– on day 11 but became MAP1A⁺/MAP2A&B⁺ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.     

Development of chicken intrafusal muscle fibers

The first sign of developing intrafusal fibers in chicken leg muscles appeared on embryonic day (E) 13 when sensory axons contacted undifferentiated myotubes. In sections incubated with monoclonal antibodies against myosin heavy chains (MHC) diverse immunostaining was observed within the developing intrafusal fiber bundle. Large primary intrafusal myotubes immunostained moderately to strongly for embryonic and neonatal MHC, but they were unreactive or reacted only weakly with antibodies against slow MHC. Smaller, secondary intrafusal myotubes reacted only weakly to moderately for embryonic and neonatal MHC, but 1–2 days after their formation they reacted strongly for slow and slow-tonic MHC. In contrast to mammals, slow-tonic MHC was also observed in extrafusal fibers. Intrafusal fibers derived from primary myotubes acquired fast MHC and retained at least a moderate level of embryonic MHC. On the other hand, intrafusal fibers developing from secondary myotubes lost the embryonic and neonatal isoforms prior to hatching and became slow. Based on relative amounts of embryonic, neonatal and slow MHC future fast and slow intrafusal fibers could be first identified at E14. At the polar regions of intrafusal fibers positions of nerve endings and acetylcholinesterase activity were seen to match as early as E16. Approximately equal numbers of slow and fast intrafusal fibers formed prenatally; however, in postnatal muscle spindles fast fibers were usually in the majority, suggesting that some fibers transformed from slow to fast.     

Nitric oxide-induced carbonylation of Bcl-2, GAPDH and ANT precedes apoptotic events in insulin-secreting RINm5F cells

Generation of high levels of nitric oxide (NO) following induction of NOS2 by interleukin-1 beta (IL-1beta) triggers beta cell apoptosis in insulin-secreting RINm5F cells. Mitochondrial and nuclear events such as downregulation of the antiapoptotic protein Bcl-2, activation of the pore responsible for the permeability transition (PT) and DNA fragmentation are involved in the process. We report in the present paper that exposure of insulin-producing RINm5F cells to NO donors and to IL-1beta leads to oxidative carbonylation of both Bcl-2 and the adenine nucleotide translocator (ANT) component of the mitochondrial PT pore. When the effect of endogenous generation of high concentrations of NO following exposure of cells to IL-1beta was studied, carbonylation of Bcl-2 preceded downregulation of the protein. Overexpression of Mn-SOD decreases substantially the extent of Bcl-2 carbonylation in SIN-1-exposed cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibition, carbonylation and translocation from cytoplasm to nucleus and DNA fragmentation were also induced by DETA/NO exposure. DETA/NO-induced carbonylation of Bcl-2 and ANT proteins takes place 6 h before apoptotic release of histone-associated DNA to cytoplasm. Time course studies also reveal a close parallel between GAPDH translocation to nucleus and carbonylation. Inhibitors of lipooxidation end products formation such as piridoxamine (PM) and aminoguanidine (AG) block NO-triggered carbonylation of Bcl-2, ANT and GAPDH, prevent NO-induced GAPDH enzyme inhibition and nuclear translocation and DNA fragmentation. Our results support the notion that the oxidative carbonylation of proteins plays a role in the control of NO-induced apoptosis.     

Shotgun proteome analysis of protein cleavage in apoptotic cells

A new shotgun proteomics approach was employed to identify degraded proteins. Jurkat T-cells were induced to undergo apoptosis by Fas (CD95/Apo-1) stimulation. The proteins were separated by large (30 cm) sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by liquid chromatography-tandem mass spectrometry after digestion of 100 gel slices with trypsin. The molecular masses of the individual gel slices were calculated through the known theoretical masses of the identified proteins. Proteins were defined as degradation candidates if either the empirical determined molecular mass was at most 80% of the theoretical value, or if proteins were identified in clearly different gel slices. In this manner, the degradation of 11 already identified apoptosis-modified proteins was confirmed and nine until now unknown degradation candidate proteins identified. Degradation during apoptosis must be verified by additional techniques such as in vitro caspase assays as shown for nucleolin and Rho GDI 2. The results presented confirm the suitability of a shotgun approach for the identification of putative protease targets.     

Distribution of GABAergic perikarya and terminals in the centers of the higher auditory pathway of the chicken

Summary The distribution of presumed GABAergic neurons and axon terminals in nuclei of the higher auditory pathway of the chicken was investigated by immunocytochemical methods employing antisera to the rate-limiting enzyme of GABA synthesis, glutamic acid decarboxylase, and to GABA. In the mesencephalic auditory center (MLD) about 20% of the cells reveal immunoreactivity. In contrast, the thalamic relay station nucleus ovoidalis is devoid of immunostained somata. This nucleus contains a high density of punctate immunoreactive structures presumed to be GABAergic axon terminals. In the auditory forebrain center field L and the auditory portions of the hyperstriatum ventrale, up to 8% of the cells were immunopositive. These neurons were significantly smaller than estimated from measurements of the overall cell population in these nuclei. From the two-dimensional arrangement of immunopositive neurons it is suggested that the GABAergic system in the avian auditory telencephalon consists of two separate groups of neurons: one subgroup mediating local inhibitory interactions, the other responsible for lateral inhibition between different frequency representations.This work was supported by the Deutsche Forschungsgemeinschaft (SFB 45)     

Differential expression of the GABA transporters GAT-1 and GAT-3 in brains of rats,cats, monkeys and humans

The homeostasis of GABA is critical to normal brain function. Extracellular levels of GABA are regulated mainly by plasmalemmal gamma-aminobutyric acid (GABA) transporters. Whereas the expression of GABA transporters has been extensively studied in rodents, validation of this data in other species, including humans, has been limited. As this information is crucial for our understanding of therapeutic options in human diseases such as epilepsy, we have compared, by immunocytochemistry, the distributions of the GABA transporters GAT-1 and GAT-3 in rats, cats, monkeys and humans. We demonstrate subtle differences between the results reported in the literature and our results, such as the predominance of GAT-1 labelling in neurons rather than astrocytes in the rat cortex. We note that the optimal localisation of GAT-1 in cats, monkeys and humans requires the use of an antibody against the human sequence carboxyl terminal region of GAT-1 rather than against the slightly different rat sequence. We demonstrate that GAT-3 is localised mainly to astrocytes in hindbrain and midbrain regions of rat brains. However, in species such as cats, monkeys and humans, additional strong immunolabelling of oligodendrocytes has also been observed. We suggest that differences in GAT distribution, especially the expression of GAT-3 by oligodendrocytes in humans, must be accommodated in extrapolating rodent models of GABA homeostasis to humans.Grant support was provided by the National Health and Medical Research Council (Australia) grant nos. 210127 and 102448, and a Senior Research Fellowship to David Pow.